Ponce-Coria José, San-Cristobal Pedro, Kahle Kristopher T, Vazquez Norma, Pacheco-Alvarez Diana, de Los Heros Paola, Juárez Patricia, Muñoz Eva, Michel Gabriela, Bobadilla Norma A, Gimenez Ignacio, Lifton Richard P, Hebert Steven C, Gamba Gerardo
Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Tlalpan, 14000 Mexico City, Mexico.
Proc Natl Acad Sci U S A. 2008 Jun 17;105(24):8458-63. doi: 10.1073/pnas.0802966105. Epub 2008 Jun 11.
The Na(+):K(+):2Cl(-) cotransporter (NKCC2) is the target of loop diuretics and is mutated in Bartter's syndrome, a heterogeneous autosomal recessive disease that impairs salt reabsorption in the kidney's thick ascending limb (TAL). Despite the importance of this cation/chloride cotransporter (CCC), the mechanisms that underlie its regulation are largely unknown. Here, we show that intracellular chloride depletion in Xenopus laevis oocytes, achieved by either coexpression of the K-Cl cotransporter KCC2 or low-chloride hypotonic stress, activates NKCC2 by promoting the phosphorylation of three highly conserved threonines (96, 101, and 111) in the amino terminus. Elimination of these residues renders NKCC2 unresponsive to reductions of Cl(-). The chloride-sensitive activation of NKCC2 requires the interaction of two serine-threonine kinases, WNK3 (related to WNK1 and WNK4, genes mutated in a Mendelian form of hypertension) and SPAK (a Ste20-type kinase known to interact with and phosphorylate other CCCs). WNK3 is positioned upstream of SPAK and appears to be the chloride-sensitive kinase. Elimination of WNK3's unique SPAK-binding motif prevents its activation of NKCC2, as does the mutation of threonines 96, 101, and 111. A catalytically inactive WNK3 mutant also completely prevents NKCC2 activation by intracellular chloride depletion. Together these data reveal a chloride-sensing mechanism that regulates NKCC2 and provide insight into how increases in the level of intracellular chloride in TAL cells, as seen in certain pathological states, could drastically impair renal salt reabsorption.
钠-钾-2氯协同转运蛋白（NKCC2）是袢利尿剂的作用靶点，在巴特综合征中发生突变，巴特综合征是一种异质性常染色体隐性疾病，会损害肾脏髓袢升支粗段（TAL）的盐重吸收。尽管这种阳离子/氯协同转运蛋白（CCC）很重要，但其调节机制在很大程度上仍不清楚。在这里，我们表明，通过共表达钾-氯协同转运蛋白KCC2或低氯低渗应激实现非洲爪蟾卵母细胞内的氯化物耗竭，可通过促进氨基末端三个高度保守的苏氨酸（96、101和111）的磷酸化来激活NKCC2。消除这些残基会使NKCC2对细胞内[Cl⁻]的降低无反应。NKCC2的氯化物敏感性激活需要两种丝氨酸-苏氨酸激酶WNK3（与WNK1和WNK4相关，这两个基因在一种孟德尔形式的高血压中发生突变）和SPAK（一种已知与其他CCC相互作用并使其磷酸化的Ste20型激酶）的相互作用。WNK3位于SPAK的上游，似乎是氯化物敏感激酶。消除WNK3独特的SPAK结合基序会阻止其对NKCC2的激活，苏氨酸96、101和111的突变也会如此。催化无活性的WNK3突变体也完全阻止细胞内氯化物耗竭对NKCC2的激活。这些数据共同揭示了一种调节NKCC2的氯化物传感机制，并深入了解了在某些病理状态下TAL细胞内氯化物水平升高如何严重损害肾脏盐重吸收。
